Phenotypic and Draft Genome Sequence Analyses of a Paenibacillus sp. Isolated from the Gastrointestinal Tract of a North American Gray Wolf (Canis lupus)

Round 1

Reviewer 1 Report

Dear authors

Please, find the attached file.

Comments for author File: Comments.pdf

Moderate editing of English language required

Author Response

Reply

Reviewer 1 stated that we do not have a representative title for the manuscript. However, no alternative title was suggested, and the title is typical of these types of reports for characterizing bacterial isolates. One can point to several publications in the literature such as:

https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-023-02939-1 and

https://www.frontiersin.org/articles/10.3389/fmicb.2023.1179857/full

Consequently, we would like to keep the current title unless the editors have a specific suggestion as how to edit the title. We did add a descriptor to the ‘Gray Wolf’ in the title.

The request as to how the growth inhibition occurs is beyond the scope of a brief communication. We followed the protocol as reported by Hardy et al., 2020:

https://www.frontiersin.org/articles/10.3389/fmicb.2019.02977/full

In this publication the goal was to identify individual bacterial species that possess anti-S. aureus activity. The inhibitory activity was not investigated as this entails extensive biochemical analyses to determine mode of action that could include a variety of mechanisms including bacteriocins, etc.

The issue of treatment options for canine inflammatory bowel disease (cIBD) is an ongoing issue. The reviewer asked, “How no treatment options?” (L38-40) and a sentence was added stating that treatments have included corticosteroids, high dietary fiber, and probiotics. However, these treatments are unsuccessful in curing cIBD

We were requested to add text as to the properties and mode of action of probiotics (L 60, now 64-66). This has been elaborated on in the introduction section as referenced in [19].

The reviewers requested we reference a preliminary study and expand upon our reasoning for the investigations (L65-66). The term “preliminary” was eliminated and a sentence was added to explain the reasoning behind our investigations (L68-71).

We altered the text in L68 (now L75-76) to GI tract material from the small intestine and added the fact that the wolf had been killed in an automobile accident and had been dead for only a day, essentially the time in which it took to transport the carcass to the veterinary diagnostic laboratory. In L77 we report that the media types are routinely used for fastidious bacterial organisms. Also, a basis for choosing bacterial isolates to obtain genome sequence was a positive result by growth inhibition of target bacteria (L85-87). We have also reported the concentration of the target bacteria (L103)

We edited the text in L122-124 to eliminate repetitive information and have defined the acronym for BBHK in the methods (now L85) section and expanded the criteria for choosing isolates for further characterization. Basically, students chose isolates based on unique colony formation (L125).

A more thorough explanation of the methods that are reported in the results (L133-135) is now presented in the methods section (L92-96). MIO media, Simmons citrate or dextrose media, maltose, lactose, and dextrose growth assays are cited in the Methods. Also, the antibiotic sensitivities assay by disc diffusion is included in the methods.

In L142-145 the Reviewer asked for a control and the size of the clearance zone demonstrating growth inhibition by the ClWae2A. We have strengthened that portion of the text stating that the control is a media-soaked disc that produced no clearance zone, and the area of clearance is 2mm (now L161-163).

The reviewer stated that the Discussion was “poor” and consequently, we have expanded the discussion. This includes addressing important noting attributes for new canine probiotics (L253-258). Also, it should be noted that overall diversity of the microbiome of wolves versus dogs have been studied (refs. 40-42), but no individual species of bacteria have been isolated that have been clearly identified as being different from one or the other species. Reviewer two requested additional references and only one was located (now refs. 52,53) that addresses this issue. To our knowledge this is the first report an axenic culture of a bacterium from a North American Gray wolf.

Reviewer 2 Report

This reviewer agreed that the methods of bacterial analysis and other methods used in this study were well planned.

The following statement can improve the manuscript by adding the following information.

L68-70: How many months have passed since the wolve’s death ? Were the dead wolf cryopreserved?

In Discussion: Are the bacterial species isolated in this study absent from domesticated dogs and wolves? Additional references are requested.

Author Response

Reply:

Reviewer 2 asked L68-70: How many months have passed since the wolve’s death ? Were (was) the dead wolf cryopreserved?

The animal had been dead for one day and this is reported in L75-76. The animal itself was unable to be cryopreserved as it is beyond the capabilities of the veterinary diagnostic laboratory to cryopreserve every animal that is necropsied at the facility.

Reviewer 3 Report

Phenotypic and draft genome sequence analyses of a Paenibacillus sp. isolated from a grey wolf (Canis lupus) gastrointestinal tract.

Dear Authors,

the manuscript is interesting and quite well described, but in my opinion the structure of the article needs correction. Some sentences from the Results better suited to the Materials and Methods subsection and the first part of the Conclusions better fitted to the Discussion. The number of replications may also be a problem, one gray wolf may show high individual variability within the microbiome depending on the specificity of the diet. However, the research focused more on the possibility of detecting Paenibacillus sp., but the number of samples used for the analysis would be valuable information. Below I add some suggestions helpful during revision process:

Line 68-81

The number of samples taken for analysis (phylogenic and sequencing) from the gray wolf will be valuable information for these studies, because when extending the analysis to a given population (sample from the population) in the future, the variability in the composition of the microbiome of a larger number of individuals will have to be taken into account. I am aware that in the case of wolves it is difficult to obtain more samples to collect data with the appropriate power of a test for analysis taking into account the variability of the composition of the microbiome, but perhaps in a non-invasive way it would be possible to obtain a larger number of samples from wolves kept in Rescure Stations in National Parks. In the case of this analysis, the most important aspect was the presence of appropriate groups of microorganisms, which was confirmed in the analysis. However, in the conclusions, it would be necessary to add information about the extension of studies to a larger number of animals in the future.

Line 122-129

This part seems to be better to use in the Materials and Methods subsection.

Line 206

Maybe better will be to merge subsections Results and Discussion in one as a: 3. Results and discussion, and then change next subsection as: 4. Conclusions.

Line 232-238

The References are not included in the conclusions. Only summarizing the entire manuscript in short sentences should be included. Better will be to move this initial part to the Discussions. For the Conclusions, you can leave lines 238-242 and expand it a bit without citing other references, based on the results of preliminary research.

Line 274

The References:

·       Bold must be used in case year of publication,

·       Dots must be used in abbreviation of Journal’s title: 1 (Nat. Microbiol.), 4, 8, 9, 11, 12, 15, 17, 19, 20, 21, 25, 28, 31, 32, 33, 41, 42, 43, 45, 47, 48, 50, 51, 52, 54,

Line 351 and 365

Nucleic Acids Res. - italics needed

Line 368 and 370

Bioinformatics - italics needed

Line 394

BMC Res. Notes – italics needed

Line 397

Antibiotics – italics needed

Author Response

Reply:

Reviewer 3 asked if The number of samples taken for analysis (phylogenic and sequencing) from the gray wolf will be valuable information for these studies We have included a statement that a total of 25 axenic bacterial isolates were obtained and that a single isolate (l. 86-87) was chosen for whole genome sequence as described in the results (L140-142)

The Reviewer also asked if we could obtain more samples from free-ranging animals and requested that we add information about the extension of studies to a larger number of animals in the future. However, we are not in a position to obtain more wolf GI tract bacterial isolates due to financial constraints as funding for the undergraduate research is limited to characterizing current isolates.

In Line 122-129, this part seems to be better to use in the Materials and Methods subsection. We agree, and this text has been eliminated from the results

Also, Line 206 May be better to merge subsections Results and Discussion in one as a: 3. Results and discussion, and then change next subsection as: 4. Conclusions. Although this is a valuable comment, we would like to keep the discussion section separate from the results as dictated by the journal template for manuscripts.

Line 232-238 The References are not included in the conclusions. Only summarizing the entire manuscript in short sentences should be included. Better will be to move this initial part to the Discussions. For the Conclusions, you can leave lines 238-242 and expand it a bit without citing other references, based on the results of preliminary research. As directed by the reviewer this portion of the text has been edited such that the text is now in the Discussion section with a brief conclusion statement (L256-265).

All issues with the references have been corrected and incorporated into the manuscript. Sincere apologies for the oversight.

Round 2

Reviewer 3 Report

Thank you for revision process. I don't have more suggestions.